Genome-Wide Identification, Characterization, and Expression of TCP Genes Family in Orchardgrass

Plant-specific TCP transcription factors regulate several plant growth and development processes. Nevertheless, little information is available about the TCP family in orchardgrass (Dactylis glomerata L.). This study identified 22 DgTCP transcription factors in orchardgrass and determined their structure, phylogeny, and expression in different tissues and developmental stages. The phylogenetic tree classified the DgTCP gene family into two main subfamilies, including class I and II supported by the exon–intron structure and conserved motifs. The DgTCP promoter regions contained various cis-elements associated with hormones, growth and development, and stress responses, including MBS (drought inducibility), circadian (circadian rhythms), and TCA-element (salicylic acid responsiveness). Moreover, DgTCP9 possibly regulates tillering and flowering time. Additionally, several stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, indicting their potential effects regarding regulating responses to the respective stress. This research offers a valuable basis for further studies of the TCP gene family in other Gramineae and reveals new ideas for increasing gene utilization.


Introduction
The plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family was first discovered in 1999 [1]. The acronym TCP comes from four genes in three species: T (TB1 from maize [Zea mays]) [2], C (CYC from snapdragon [Antirrhinum majus]) [3], and P (PCFs from rice [Oryza sativa]) [4]. Members of the TCP family have 59 amino acids and an atypical basic helix-loop-helix (bHLH) motif at the N-terminus called the TCP domain which is responsible for protein-protein interactions, nuclear protein localization, and DNA binding [1,4]. Moreover, the TCP domain classifies these proteins into two groups: class I and II [5]. The clearest distinction between class I and II is the basic region of the TCP domain where class I members lost four amino acids. Class II is divided into CYC/TB1 and CIN subclades [5,6]. Arginine-rich motifs with 18-20 residues (R domain) are usually found in class II [1].

Gene Structure and Motif Analysis
The exon-intron structures of DgTCPs were generated based on available genomic information and coding sequences from the Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn/, accessed on 6 May 2022) [52]. The online Multiple Expectation Maximization for Motif Elicitation (MEME) software (http://meme-suite.org/, accessed on 11 May 2022) was used to identify the conserved DgTCP proteins motifs (considering ten maximum motifs and default settings) [53].

Putative Promoter Cis-Acting Element Analysis
The nucleotide sequences of DgTCPs were acquired from the orchardgrass genome database (http://orchardgrassgenome.sicau.edu.cn/, accessed on 20 January 2022). The 2000 bp region upstream of all DgTCPs was considered the promoter sequence, and the cisacting promoter elements were appraised via PlantCARE (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/, accessed on 19 June 2022) [54]. The putative cis-acting elements are classified into plant hormone responses, growth and development, and biotic and abiotic stress responses.

Expression Profiles of DgTCP Family Members
The expression patterns of TCPs in the root, stem, leaf, spike, and flower were obtained from the orchardgrass genome database (Table S1) [46]. Furthermore, the expression patterns of the floral bud developmental stages before vernalization (BV), vernalization (V), after vernalization (AV), before heading (BH), and heading (H) of the late-flowering variety "Baoxing" and early-flowering variety "Donata" were obtained from the RNA-seq data (Table S2) [57]. The RNA-seq data of the TCP genes in four tissues from varieties D20170203 (low-tillering) and AKZ-NRGR66 7 (high-tillering) were obtained from Xu et al. (Table S3) [58]. The heat maps of the expression patterns were produced using TBtools [56].

Expression of 14 Selected DgTCP Genes in qRT-PCR
The total RNA of the samples under different treatments was extracted using the Hipure HP plant RNA mini kit (Magen, Guangdong, China). First-strand cDNA was synthesized using the MonScript TM RTIII ALL-in-One Mix with dsDNase kit (Monad, Suzhou, China) following the manufacturer's instructions. Primers for the 14 DgTCPs were designed using Primer 5.0 software (Table S4), and qRT-PCR was performed using the MonAmpTM SYBR ® green qPCR Mix (Monad) on the Bio-Rad CFX96 instrument. GAPDH was the internal reference gene for normalization [43], and the relative gene expression levels were evaluated by the 2 −∆∆Ct method [59]. All qRT-PCR assays were performed with three biological and technical replicates.

Identifying TCP Genes in Orchardgrass
Twenty-two genes were retrieved from the orchardgrass genome and designated as DgTCP1-DgTCP22 based on their chromosomal positioning. The protein sequence length, CDS length, molecular weight, isoelectric point (pI), and gene location of the 22 DgTCPs are captured in Table 1 and Table S5. The smallest protein was 17,433.51(DgTCP1), and the biggest was 47,382.56 kDa (DgTCP16). The pI ranged from 5.09 (DgTCP7) to 9.92 (DgTCP11), and the protein lengths were 165 (DgTCP17) to 454 (DgTCP6) aa.

Phylogeny and Classification of the DgTCP Proteins
Based on the phylogenetic tree of 22 orchardgrass, 22 rice, 21 B distachyon, and 24 A thaliana, TCP proteins were constructed using the neighbor-joining (NJ) method to clarify the phylogenetic relationships and evolutionary history of the TCP gene family  Figure 1). Two classical subfamilies, class I and class II, were identified from the topology of the NJ tree and A thaliana classification. Eleven DgTCPs belong to class I (PCF or TCP-P), and the other 11 belong to class II (TCP-C) ( Figure 1). The class II group is further divided into CYC/TB1 (4 DgTCPs) and CIN subclasses (7 DgTCPs) ( Figure 1). Thus, we performed multiple sequence alignments on the TCP domains of all DgTCP members to comprehend the phylogenetic relationships of the DgTCPs. The TCP domain comparison and phylogenetic analysis indicated that orchardgrass TCP proteins have class I (PCF) and class II (CIN and CYC/TB1) groups (Figures 1 and 2). Class I proteins lack four amino acids at their basic domain compared with class II proteins.
Based on the phylogenetic tree of 22 orchardgrass, 22 rice, 21 B distachyon thaliana, TCP proteins were constructed using the neighbor-joining (NJ) method the phylogenetic relationships and evolutionary history of the TCP gene family Two classical subfamilies, class I and class II, were identified from the topology tree and A thaliana classification. Eleven DgTCPs belong to class I (PCF or TCP-P other 11 belong to class II (TCP-C) ( Figure 1). The class II group is further div CYC/TB1 (4 DgTCPs) and CIN subclasses (7 DgTCPs) ( Figure 1). Thus, we perfor tiple sequence alignments on the TCP domains of all DgTCP members to compr phylogenetic relationships of the DgTCPs. The TCP domain comparison and ph analysis indicated that orchardgrass TCP proteins have class I (PCF) and class II CYC/TB1) groups (Figures 1 and 2). Class I proteins lack four amino acids at t domain compared with class II proteins.  An unrooted phylogenetic tree containing TCP proteins from orchardgrass, rice, A thaliana, and B distachyon. Green shading, CIN subclass; yellow shading, CYC/TB1 subclass; purple shading, PCF subclass. The grey circle, red pentagram, black square, and blue triangle represent the rice, orchardgrass, A thaliana, and B distachyon TCPs, respectively.

The DgTCP Gene Structure and Protein Motif
The structural characteristics of all DgTCPs were analyzed to comprehend the evolution of the TCP gene family in orchardgrasss ( Figure 3b). All class I DgTCP genes, except DgTCP5 and DgTCP22, lack introns. In class II, all CIN genes possess one or two introns, while CYC/TB1 genes lack introns. Genes 2023, 14, x FOR PEER REVIEW 6 of 20 Figure 2. The logo and sequence alignment of TCP domains from orchardgrass. The basic helixloop-helix structure has been marked.

The DgTCP Gene Structure and Protein Motif
The structural characteristics of all DgTCPs were analyzed to comprehend the evolution of the TCP gene family in orchardgrasss ( Figure 3b). All class I DgTCP genes, except DgTCP5 and DgTCP22, lack introns. In class II, all CIN genes possess one or two introns, while CYC/TB1 genes lack introns. Figure 3c shows 10 conserved motifs of the 22 DgTCP proteins which were identified using MEME to reveal the structural characteristics of orchardgrass TCP. The amino acid sequence for each motif (Table S6) shows that the conserved motifs have 6-42 amino acids. All DgTCP protein contain motifs 1 and 2. Moreover, DgTCP proteins from the same subfamilies contain similar motifs. For instance, members of clade PCF contain motif 3, while the clades CIN and CYC/TB1 lack motif 3. All clade CIN members contain motifs 7 and 9, while PCF members contain motifs 5, 6, and 8.
Additionally, some motifs, such as motifs 4 and 10, are shared by two classes. These results indicate that the motifs present only in some subgroups may be associated with unique functions. Nevertheless, the unique functions of these motifs in the plant life cycle have not been identified and need to be explored further.

Chromosomal Localization, Gene Duplication, and Synteny Analysis
The 22 orchardgrass TCP genes are randomly distributed on 7 chromosomes ( Figure  4). Chromosome 4 contained six TCP genes, and chromosomes 3 and 5 had five TCP genes each. Three TCP genes mapped to chromosome 1, but only one mapped to chromosomes 2, 6, and 7.
The duplication event is important for analyzing the evolution and expansion of the gene families. The orchardgrass genome has five pairs of segmental duplicates (Table S7) (Table S6) shows that the conserved motifs have 6-42 amino acids. All DgTCP protein contain motifs 1 and 2. Moreover, DgTCP proteins from the same subfamilies contain similar motifs. For instance, members of clade PCF contain motif 3, while the clades CIN and CYC/TB1 lack motif 3. All clade CIN members contain motifs 7 and 9, while PCF members contain motifs 5, 6, and 8.
Additionally, some motifs, such as motifs 4 and 10, are shared by two classes. These results indicate that the motifs present only in some subgroups may be associated with unique functions. Nevertheless, the unique functions of these motifs in the plant life cycle have not been identified and need to be explored further.

Chromosomal Localization, Gene Duplication, and Synteny Analysis
The 22 orchardgrass TCP genes are randomly distributed on 7 chromosomes (Figure 4). Chromosome 4 contained six TCP genes, and chromosomes 3 and 5 had five TCP genes each. Three TCP genes mapped to chromosome 1, but only one mapped to chromosomes 2, 6, and 7.
The duplication event is important for analyzing the evolution and expansion of the gene families. The orchardgrass genome has five pairs of segmental duplicates (Table S7)

Chromosomal Localization, Gene Duplication, and Synteny Analysis
The 22 orchardgrass TCP genes are randomly distributed on 7 chromosomes ( Figure  4). Chromosome 4 contained six TCP genes, and chromosomes 3 and 5 had five TCP genes each. Three TCP genes mapped to chromosome 1, but only one mapped to chromosomes 2, 6, and 7.
The duplication event is important for analyzing the evolution and expansion of the gene families. The orchardgrass genome has five pairs of segmental duplicates (Table S7) (Table  S8). These results indicate that the TCPs in the monocotyledons are highly conserved and homologous.

Putative Cis-Acting Elements of Orchardgrass DgTCPs
The cis-elements in promoters are essential for transcriptional regulation and ge function analysis. Therefore, to provide further insight into the gene functions and reg lation mechanisms of DgTCP genes, 93 cis-elements possibly involved in phytohormo response, plant growth and development, and stress response were identified to unra the functions and regulatory mechanisms of DgTCP genes (Table S9). The TATA-a CAAT-box had the most cis-elements among the 22 DgTCPs (Table S9). Interestingly, t AACA-motif, MBSI, HD-Zip 1, circadian, and AuxRR-core only existed in 1 of 22 DgTC (Figure 7), indicating their likely unique roles in those genes and, by extension, the reg latory pathways and processes involving those genes.
The promoter regions of one and three DgTCPs are two cis-elements (AACA-mo and GCN4-motif) that participate in endosperm expression. Besides, NON-box and CA box were associated with meristem expression plant growth and development. The see specific regulatory element (RY element) and zein metabolism regulatory element ( site) were identified in six and nine DgTCPs, respectively. In addition, a circadian cont

Putative Cis-Acting Elements of Orchardgrass DgTCPs
The cis-elements in promoters are essential for transcriptional regulation and gene function analysis. Therefore, to provide further insight into the gene functions and regulation mechanisms of DgTCP genes, 93 cis-elements possibly involved in phytohormone response, plant growth and development, and stress response were identified to unravel the functions and regulatory mechanisms of DgTCP genes (Table S9). The TATA-and CAAT-box had the most cis-elements among the 22 DgTCPs (Table S9). Interestingly, the AACA-motif, MBSI, HD-Zip 1, circadian, and AuxRR-core only existed in 1 of 22 DgTCPs (Figure 7), indicating their likely unique roles in those genes and, by extension, the regulatory pathways and processes involving those genes. The promoter regions of one and three DgTCPs are two cis-elements (AACA-motif and GCN4-motif) that participate in endosperm expression. Besides, NON-box and CAT-box were associated with meristem expression plant growth and development. The seed-specific regulatory element (RY element) and zein metabolism regulatory element (O2 site) were identified in six and nine DgTCPs, respectively. In addition, a circadian control element (circadian), a flavonoid biosynthetic regulation element (MBSI), a cell cycle regulation element (MSA-like), and a palisade mesophyll cells regulatory element (HD-Zip 1) were also discovered in the promoter regions of DgTCPs (Figure 7). In several hormone-related cis-elements, the salicylic acid (TCA element), the auxin-responsive element (AuxRR core and TGA element), the gibberellin-responsive element (GARE motif, TATC-box, and P-box), the Me-JA-responsive element (CGTCA motif and TGACG motif), and the ABA-responsive element (ABRE) were found in the promoter region of 9, 9, 15, 19, and 19 DgTCP genes, respectively (Figure 7). In addition, the DgTCP promoters contained several cis-elements that were related to several stresses (drought, anaerobic induction, and low temperature) (Figure 7).

Expression Profiles of DgTCPs in Different Tissues and Developmental Stages
The expression profiles of 2, 5, and 14 DgTCP genes were the highest in the leaf, spike, and stem, respectively ( Figure 8a). Moreover, DgTCP1 and DgTCP9 had higher transcription levels in the flowers, revealing that these genes might be important for the growth of different orchardgrass tissues.
The expression patterns of the early-(Baoxing) and late-flowering (Donata) cultivars were analyzed at five flower bud development stages to identify their potential physiological functions in flowering. In most developmental stages, the expression of DgTCP9 and DgTCP6 was higher in "Baoxing" than "Donata" (Figure 8b). TCP15 expression was similar in "Baoxing" and "Donata" before, during (downregulated), and after vernalization (upregulated). TCP18 was significantly upregulated during vernalization in "Baoxing" but showed no change in "Donata". However, it was upregulated in "Baoxing" and "Donata" during the late vernalization stage and similar in "Baoxing" and "Donata". From after vernalization to the heading stage, DgTCP2, DgTCP4, DgTCP16, DgTCP17, and DgTCP21 were significantly upregulated during the before heading stage in "Baoxing" and the heading stage in "Donata".

Expression Profiles of DgTCPs in Different Tissues and Developmental Stages
The expression profiles of 2, 5, and 14 DgTCP genes were the highest in the leaf, spike, and stem, respectively ( Figure 8a). Moreover, DgTCP1 and DgTCP9 had higher transcription levels in the flowers, revealing that these genes might be important for the growth of different orchardgrass tissues.
The expression patterns of the early-(Baoxing) and late-flowering (Donata) cultivars were analyzed at five flower bud development stages to identify their potential physiological functions in flowering. In most developmental stages, the expression of DgTCP9 and DgTCP6 was higher in "Baoxing" than "Donata" (Figure 8b). TCP15 expression was similar in "Baoxing" and "Donata" before, during (downregulated), and after vernalization (upregulated). TCP18 was significantly upregulated during vernalization in "Baoxing" but showed no change in "Donata". However, it was upregulated in "Baoxing" and "Donata" during the late vernalization stage and similar in "Baoxing" and "Donata". From after vernalization to the heading stage, DgTCP2, DgTCP4, DgTCP16, DgTCP17, and DgTCP21 were significantly upregulated during the before heading stage in "Baoxing" and the heading stage in "Donata". Gene expression data were retrieved from four tissues of low-(D20170203) and hightillering (AKZ-NRGR667) orchardgrass to determine the roles of DgTCPs in regulating growth, and development. All DgTCPs were differentially expressed in the four tissues, contrary to their expression under normal conditions (Figure 9). In tiller buds, over half Gene expression data were retrieved from four tissues of low-(D20170203) and hightillering (AKZ-NRGR667) orchardgrass to determine the roles of DgTCPs in regulating growth, and development. All DgTCPs were differentially expressed in the four tissues, contrary to their expression under normal conditions (Figure 9). In tiller buds, over half of the DgTCPs were highly expressed in the low-(D20170203) and high-tillering (AKZ-NRGR667) varieties. The expression of TCP1 was significantly higher in D20170203 than AKZ-NRGR667 in the four tissues. However, the expression of TCP20 was higher in the leaves of AKZ-NRGR667 than in D20170203 but similar in other tissues. The expression of TCP10 was higher in the tiller bud of D20170203 than in AKZ-NRGR667 but similar in other tissues. Interestingly, the expression of TCP9 was higher in the tiller buds of D20170203 than those of AKZ-NRGR667 but lower in the leaves of D20170203. Thus, DgTCP1 and DgTCP10 probably inhibit tiller bud development in the two varieties in a differential pattern, ultimately resulting in different phenotypes. DgTCP1 and DgTCP10 probably inhibit tiller bud development in the two varieties in a differential pattern, ultimately resulting in different phenotypes.  Figure 10 shows the expressions of 14 DgCTPs in "Baoxing" under six abiotic stresses (drought, salt, alkali, Me-JA, ABA, and SA). Salt stress suppressed the expression of two genes (DgTCP8 and DgTCP18) throughout the salt time points and downregulated eleven genes in the early stages (1-3 h) of salt stress. Drought stress upregulated 13 DgTCPs, and the highest values ranged from 1.13-fold (DgTCP3) to 16.89-fold (DgTCP1). Additionally, six genes (DgTCP2, DgTCP12, DgTCP15, DgTCP16, DgTCP17, and DgTCP18) showed significantly higher expression 1 h after treatment with an alkali solution. In contrast, other DgTCPs were upregulated at 6 h of alkali treatment, while DgTCP8 was suppressed at all time points. Nine DgTCPs were highly induced under ABA treatment, and DgTCP12 displayed the highest expression. Nevertheless, ABA treatment inhibited DgTCP3, DgTCP8, DgTCP9, DgTCP16, and DgTCP19 at all time points. Me-JA treatment upregulated five (DgTCP1, DgTCP2, DgTCP6, DgTCP10, and DgTCP17), one (DgTCP8), and two genes (DgTCP3, DgTCP12), which peaked at 3, 6, and 12 h, respectively. These genes were upregulated ranging from 1.26-fold (DgTCP18) to 20.16-fold (DgTCP12). In contrast, Me-JA showed no observable regulation in four genes (DgTCP4, DgTCP8, DgTCP15, and DgTCP16) but downregulated DgTCP9 and DgTCP19. Furthermore, SA treatment suppressed the expression of DgTCP3, DgTCP9, and DgTCP19 across the time points, with six (DgTCP1, DgTCP2 DgTCP12, DgTCP15, DgTCP16, and DgTCP17) and three genes (DgTCP4, DgTCP6, and DgTCP18) peaking at 6 and 24 h, respectively. Six stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, but DgTCP levels varied under different stresses and time points when combined with the stress expression pattern data.  Figure 10 shows the expressions of 14 DgCTPs in "Baoxing" under six abiotic stresses (drought, salt, alkali, Me-JA, ABA, and SA). Salt stress suppressed the expression of two genes (DgTCP8 and DgTCP18) throughout the salt time points and downregulated eleven genes in the early stages (1-3 h) of salt stress. Drought stress upregulated 13 DgTCPs, and the highest values ranged from 1.13-fold (DgTCP3) to 16.89-fold (DgTCP1). Additionally, six genes (DgTCP2, DgTCP12, DgTCP15, DgTCP16, DgTCP17, and DgTCP18) showed significantly higher expression 1 h after treatment with an alkali solution. In contrast, other DgTCPs were upregulated at 6 h of alkali treatment, while DgTCP8 was suppressed at all time points. Nine DgTCPs were highly induced under ABA treatment, and DgTCP12 displayed the highest expression. Nevertheless, ABA treatment inhibited DgTCP3, DgTCP8, DgTCP9, DgTCP16, and DgTCP19 at all time points. Me-JA treatment upregulated five (DgTCP1, DgTCP2, DgTCP6, DgTCP10, and DgTCP17), one (DgTCP8), and two genes (DgTCP3, DgTCP12), which peaked at 3, 6, and 12 h, respectively. These genes were upregulated ranging from 1.26-fold (DgTCP18) to 20.16-fold (DgTCP12). In contrast, Me-JA showed no observable regulation in four genes (DgTCP4, DgTCP8, DgTCP15, and DgTCP16) but downregulated DgTCP9 and DgTCP19. Furthermore, SA treatment sup-pressed the expression of DgTCP3, DgTCP9, and DgTCP19 across the time points, with six (DgTCP1, DgTCP2 DgTCP12, DgTCP15, DgTCP16, and DgTCP17) and three genes (DgTCP4, DgTCP6, and DgTCP18) peaking at 6 and 24 h, respectively. Six stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, but DgTCP levels varied under different stresses and time points when combined with the stress expression pattern data.

Discussion
The plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family are crucial plant-specific transcription factors with various functions in many processes, including hormone biosynthesis, flower development, leaf development, lateral branching, and defence response. To date, the TCP gene family has been identified in many plants, such as switchgrass (Panicum virgatum) [60], maize [61], Arabidopsis, and rice [62]. However, a comprehensive report of the TCP gene family in high-quality forages, such orchardgrass, is lacking. This research identified 22 TCP genes in the orchardgrass genome [46]. All the corresponding DgTCP proteins have a highly conserved TCP domain (motifs 1 and 2). Thus, DgTCPs possibly have similar DNA-binding and protein-protein interaction patterns [1,4]. Moreover, sequence alignment and phylogenetic analysis revealed that the 22 DgTCPs are divided into two major subclasses (Figures 2 and 3), which is consistent with previous results [5]. Each subclass contained TCP genes from B distachyon, rice, and Arabidopsis. Furthermore, the orchardgrass TCP genes are closely related to the TCPs of rice and B distachyon, indicating that they evolved from a common ancestor as Gramineae. These results show that many TCP genes from the same ancestor possibly experienced different differentiation patterns at different lineages. Moreover, DgTCPs in the same class and subclass had similar exon-intron structures ( Figure 3b) and relatively conserved motifs (Figure 3c), further supporting the close evolutionary relationships between DgTCPs.
Abiotic stresses affect plant growth and development, quality, and yield [33], and TCP genes are broadly involved in the regulatory processes of plant life [71]. Therefore, exploring the potential functions of TCP genes in orchardgrass under different abiotic stresses is necessary. In this study, salt and drought treatments upregulated more than half of the identified DgTCPs, similar to the results from rapeseed (Brassica napus) [72], cotton [17], and switchgrass [60]. Nonetheless, ABA treatment inhibited five DgTCPs at all time points. The ABA signal transduction pathway is significant for stress response [73].
Moreover, TCPs interact with other genes in JA biosynthesis to influence growth, development, and abiotic stress responses. For instance, TCP4 encodes the enzyme that catalyzes a crucial step in JA synthesis by positively regulating the LOX2 gene [9]. Deactivating TCP4 in plants downregulates LOX2, thus reducing JA synthesis and increasing plant sensitivity to stress [9]. The expression patterns of orchardgrass TCP genes were diverse after Me-JA treatment ( Figure 10). For example, Me-JA treatment lowered the expression of DgTCP16 of the AtTCP4-like gene, which may be because the promoter region of TCP16 lacks Me-JA-related cis-elements (Figure 7).
In A thaliana, TCP8 and TCP9 combine to the TCP-promoter binding site of the SA biosynthesis gene ICS1, thus enhancing ICS1 expression [74]. Additionally, SA treatment increased the expression of many cis-elements related to SA in the promoter regions of the DgTCP genes (Figure 7), including DgTCP2, DgTCP4, DgTCP12, and DgTCP18 ( Figure 10). Therefore, DgTCP genes may play a role in SA transduction. These results suggest that DgTCP genes are essential for plants to cope with abiotic stress.
Gene function can be inferred from the expression profile of that gene [75]. Thus, this research inferred the functions of 22 DgTCPs using their expression patterns in five tissues (Figure 8a). The results showed different expression profiles of the 22 DgTCPs in five tissues, indicating that orchardgrass TCPs might be related to the development of different tissues. The highest expression of several DgTCPs, such as DgTCP3, DgTCP4, DgTCP15, and DgTCP19, occurred in the stem. Several TCP genes are highly expressed in the stem, including 60 in cotton [17], 11 in rapeseed [72], and 9 in soybean [67]. Some DgTCP genes, such as DgTCP1 and DgTCP9, are highly expressed in flowers, indicating that DgTCP genes possibly participate in flowering. As with this study, 16 and 12 highly expressed TCP genes were reported in rapeseed and soybean [67,72]. Moreover, most duplicate gene pairs had the same functions and similar expression patterns, except DgTCP4, DgTCP9, DgTCP7, and DgTCP22, which showed different expression profiles. This diverse expression may be related to differences in the evolution of duplicate genes or upstream regulatory mechanisms, causing functionalization in one of the duplicate genes.
Next, we compared the potential role of TCP genes in regulating flowering time at the five stages of late-and early-flowering orchardgrass (Figure 8b). Flowering is crucial for the growth and development of Gramineae, and variations in flowering time directly affect orchardgrass quality and yield. Moreover, vernalization is a critical way to control flowering time and floral organ development [57]. Thus, the unique expression profiles of DgTCP15 and DgTCP18 at different floral bud developmental stages suggests that these genes may regulate flowering time through the vernalization pathway. For example, A thaliana plants that overexpress AtTCP23 have the late-flowering phenotype [76]. In this study, DgTCP6 was expressed at higher levels in "Donata" than in "Baoxing" from the before vernalization stage to the heading stage of late-flowering "Donata" and earlyflowering "Baoxing". DgTCP6 and AtTCP23 belong to the same branch on the evolutionary tree, indicating that they are homologous genes. This alignment implies that DgTCP6 and AtTCP23 have similar functions. Thus, a high expression of DgTCP6 promotes the late-flowering phenotype in "Donata". Furthermore, the DgTCP9 gene, which has a similar expression pattern as DgTCP6 (Figure 8b), may also have a similar flowering regulatory function. Moreover, AtTCP4 and AtTCP13, AtTCP7 induce early flowering by directly acting on the AP1 promoter to improve its transcript activation ability and activating the transcription expression of the flowering integration gene SOC1, respectively [77,78]. The three TCPs were grouped with DgTCP16 and DgTCP21, DgTCP17, respectively ( Figure 1). Additionally, from the before heading stage to the heading stage, the earlier upregulation of DgTCP16, DgTCP17, and DgTCP21 in "Baoxing" relative to "Donata" indicates that they influence early flowering in "Baoxing" and reflect the functions of AtTCP4 and AtTCP13, AtTCP7 in A thaliana. Altogether, the diverse expression of DgTCPs at the five floral bud stages in the different cultivars indicates their regulatory role in orchardgrass flowering.
Finally, the roles of DgTCP1 in tillering were analyzed in the respective cultivars. Tillering is an important agronomic trait in forage crops as it determines the seed yield and aboveground biomass of forage grasses [79,80]. In this study (Figure 9), the tissue-specific expression patterns of DgTCP1 in the two forage varieties revealed that a high expression of DgTCP1 may suppress tillering. Moreover, OsTCP19, a DgTCP9 homologous gene (Figure 1), negatively regulates rice tillering by inhibiting DLT, which promotes tillering [81]. The unique expression of DgTCP9 in high-and low-tillering materials indicates that DgTCP9 possibly confers low-tillering in "D20170203". In summary, DgTCPs might be important for tiller development; thus, they require further experimental verification.

Conclusions
This study identified 22 DgTCPs from the whole genome of D glomerata. Phylogenetic characteristics divided the 22 DgTCPs into class I and II subfamilies. The study also revealed the protein sequence length, CDS length, pI, and molecular weight of the proteins predicted from the 22 DgTCP genes. Furthermore, we identified many cis-elements in the DgTCPpromoter sequences, revealing a complex regulatory network that possibly controls DgTCP genes. The 22 DgTCP genes contained five pairs of segmental repeat genes distributed on seven chromosomes, indicating that segmental duplication was the primary mechanism for DgTCP gene expansion.
Furthermore, the expression of the DgTCPs under various abiotic stresses at different stages (tiller bud and floral bud) and tissues suggested that many DgTCP genes regulate stress tolerance and development in orchardgrass. Specifically, TCP9 probably regulates flowering time, tiller number, and drought stress in D glomerata. This genome-wide analysis of orchardgrass is significant for identifying new DgTCP genes with novel functions and provides a foundation for breeding high-quality orchardgrass varieties and the functional validation of DgTCP genes in the future.